Metamorphosis in moths involves a continuous population of cells changing their synthetic activities to build new types of cuticle under the direct influence of two hormones, ecdysteroids and juvenoids. The changes that occur and our ability to manipulate them in vivo and in vitro presents a useful model for analyzing developmental events in our parasites and ourselves. We propose to learn how metamorphosis is regulated at the nomic level. To this end we shall isolate and characterize genes and transcripts for four cuticular proteins selected for their provocative spatial and temporal distributions. We plan to analyze chromatin to identify the regulatory domains that encompass these genes. Putative cis-acting regulatory regions will be located by using DNase I to map hypersensitive sites and by using dimethylsulfate (DMS) for "in vivo footprinting". Putative regulatory regions identified by these techniques will be used in gel retardation assays where the presence of regulatory proteins can be recognized since they reduce the mobility of DNA to which they are specifically bound. We shall test nuclear extracts from different anatomical regions and metamorphic stages as well as known trans-acting regulatory factors to sort out the nature (spatial, temporal, hormonal) of the information impinging on the cis-acting regulatory sequences of a single gene during the course of metamorphosis. We hope by these analyses to learn if hormones control metamorphosis by acting directly ont he genes that code for structural proteins. The moth epidermis presents an extremely favorable model for analyzing the fundamental issue of how chromatin structure is altered as a continuous population of cells plays out a development program that involves changes in the state of determination and activity for individual genes.